Trait matching in a multi‐species geographic mosaic of leafflower plants, brood pollinators, and cheaters

Abstract Trait matching between mutualistic species is usually expected to maintain mutualism, but empirical studies of trait complementarity and coadaptation in multi‐species assemblages—which represent the reality of most interactions in nature—are few. Here, we studied trait matching between the leafflower shrub Kirganelia microcarpa and three associated seed‐predatory leafflower moths (Epicephala spp.) across 16 populations. Behavioral and morphological observations suggested that two moths (E. microcarpa and E. tertiaria) acted as pollinators while a third (E. laeviclada) acted as a cheater. These species differed in ovipositor morphology but showed trait complementarity between ovipositor length and floral traits at both species level and population level, presumably as adaptations to divergent oviposition behaviors. However, this trait matching varied among populations. Comparisons of ovipositor length and floral traits among populations with different moth assemblages suggested an increase of ovary wall thickness where the locular‐ovipositing pollinator E. microcarpa and cheater E. laeviclada were present, while stylar pit depth was less in populations with the stylar pit‐ovipositing pollinator E. tertiaria. Our study indicates that trait matching between interacting partners occurs even in extremely specialized multi‐species mutualisms, and that although these responses vary, sometimes non‐intuitively, in response to different partner species. It seems that the moths can track changes in host plant tissue depth for oviposition.

Segraves, 2022; Kjellberg et al., 2001). At the same time, other mutualisms show coevolutionary arms race dynamics, in which trait mismatching is favored on one side of the interaction and trait matching is favored on the other (Anderson et al., 2010;Darwin, 1862;Nilsson, 1988). Brood pollination mutualisms may contain elements of both such dynamics because they contain both mutualistic (pollination) and antagonistic (seed predation) components (Althoff & Segraves, 2022). Despite the importance of trait matching and mismatching in mutualistic interactions, they have been primarily empirically demonstrated in specialized associations with few interacting species. However, interactions in nature are usually not one-to-one, but involve multiple interacting partners in multi-species assemblages (Hollens-Kuhr et al., 2021;Thompson, 2005). Consequently, testing trait matching between multiple partners is of great importance to understand the evolution and maintenance of mutualisms.
Not only are most mutualisms embedded in multi-species networks of mutualistic species, empirical, and theoretical studies indicate that antagonists may be common or at least important in many mutualistic systems (Genini et al., 2010;Jones et al., 2015). Thirdparty exploiters or "cheaters" have been demonstrated in many mutualisms, including non-pollinating parasites which evolved from pollinating ancestors (cheater yucca moths and cheater fig wasps; Machado et al., 2001;Pellmyr et al., 1996;Zhang et al., 2021), noncooperative symbionts in plant-rhizobia interactions (Gano-Cohen et al., 2020), and nectar-robbing from flowers by bees and birds (Irwin & Brody, 1998;Rojas-Nossa et al., 2016). Numerous studies have proposed explanations for why antagonists persist in mutualistic assemblages (Ferriere et al., 2002;Sachs & Simms, 2006), including the important observation that mutualists and antagonists may coexist in varied local assemblages in a geographic mosaic of different species at different sites (Hollens-Kuhr et al., 2021;Thompson, 2005). However, empirical studies describing the spatial pattern of interactions among multiple mutualists and cheaters, and corresponding trait matching, are lacking.
Although there is ample evidence that mutualism promotes stabilizing selection, mutualism may also influence population phenotypic differentiation and speciation depending on specificity, partner dependence and environmental context (Althoff, 2014;Zeng & Wiens, 2020).
In obligate mutualisms, partners are highly specialized and functional traits are reciprocally adapted to each other, so phenotypic differentiation presumably will greatly affect trait matching between partners leading to host shifts, partner replacement, and rapid reproductive isolation. For most insects, ovipositor (egg-laying structure) morphology is critical for both host plant use (Joy & Crespi, 2007) and copulation (Althoff, 2014), which is tied directly to reproductive fitness and reproductive isolation. For example, the ovipositor morphology of Tegeticula moths shifts from long, thin ovipositors to short, thick ovipositors, along with the shift of oviposition habit from locular oviposition to superficial oviposition, which maintains the reproductive isolation among species using the same yucca host (Althoff, 2014;Althoff et al., 2006). Furthermore, phylogenetic analyses suggested that speciation of at least 11 species of yucca moths was coupled with changes in ovipositor morphology (Althoff et al., 2006;Darwell et al., 2016).
In this study, we examine trait matching in local assemblages of mutualists and cheaters using one of the best-known specialized brood pollination mutualisms, leafflower-leafflower moth associations. Leafflower moths in the genus Epicephala (Gracillariidae) moths are specific to monoecious host plants in the family Phyllanthaceae (Kato et al., 2003;Luo et al., 2017) and have served as a model system in the study of plant-insect coevolution. Female moths visit male flowers to collect pollen grains with ciliated proboscises ( Figure S1) and deposit pollen grains on the stigmas of female flowers by inserting their proboscises into the flower's narrow stylar pit. Female moths subsequently lay eggs in the pollinated flowers and their larvae consume some of the host's seeds. Similar to yucca-yucca moth and fig-fig wasp systems, interactions between leafflowers and leafflower moths are not absolutely one-to-one: It is common for multiple Epicephala pollinators to coexist in some Phyllanthaceae species, or even coexist with cheaters, which lay eggs in the flowers but provide no pollination service (Kawakita et al., 2015;Kawakita & Kato, 2006).
The inability of cheaters to pollinate flowers is due to the loss of hairs on the surface of proboscises, which in pollinating species is used to collect and transport pollen grains (Kawakita & Kato, 2006 (Kawakita & Kato, 2004a, 2004bZhang et al., 2012), laid at the basal part of ovary by laterally cutting through calyx lobes and ovary by other moths associated with B. fruticosa and B. rostrata (Kawakita & Kato, 2004b;Zhang et al., 2012), laid above ovules by inserting the ovipositor into the stylar pit by moths associated with Glochidion acuminatum and G. coccineum (Chheang et al., 2022;Kato et al., 2003), laid inside the ovaries by ovipositing through the carpel wall beside the stylar pit by Epicephala lanceolaria (Luo et al., 2017), and laid inside the ovaries by inserting the ovipositor through the base of the stylar column by moths associated with G. temehaniense (Hembry et al., 2012). In addition, Kawakita and Kato (2016) studied the morphology of six Epicephala moths and hypothesized an association between an angular ovipositor and a behavior of laterally cutting through the ovary wall. In the present study, we investigated the Asian leafflower shrub Kirganelia microcarpa and its three associated Epicephala moths in 16 populations across southern China. We aimed to answer the three following questions: (1) What is the spatial pattern of pollinator and cheater coexistence? (2) Do ovipositor morphology and corresponding oviposition behaviors match different floral traits?
(3) Do the patterns of coexistence of pollinators and the cheater affect floral traits and trait matching between plant and pollinators? 2 | MATERIAL S AND ME THODS

| Pollination and oviposition behaviors
Our previous field observations in South China National Botanical Garden (Guangdong province), Huolushan Forest Park (Guangdong province), and Xinglong town (Hainan province) for more than 180 h in 3 years (2004)(2005)(2006) suggested different Epicephala species among populations with different oviposition behaviors (see Luo, 2006). For each moth, we observed and recorded the behaviors of inserting the proboscis into the stylar pit (pollination behaviors) and inserting the ovipositor into the stylar pit or laterally cutting through the ovary wall using the ovipositor (oviposition behaviors), and whether pollination occurred prior to oviposition. Each moth was collected after observation to identify the species based on morphology. In Epicephala, the presence of hairs on female proboscises is consistently predictive of pollination behavior (Kawakita & Kato, 2006. Consequently, we examined proboscis morphology to determine whether it was consistent with observed behavior: If the moth acts as a pollinator (with hairs) or cheater (without hairs).
To do this, moth heads were removed and stored in a centrifuge tube with 10% KOH solution in a water bath heated at 75°C for 12 min, stained with Eosin dye solution, and mounted in Euparal (Waldeck) on glass slides under cover slips (Kawakita et al., 2015). The slides were then examined for the presence or absence of hairs on proboscises ( Figure 2) under a stereoscope (Leica EZ4W).

| Moth rearing and trait measurements
To investigate the spatial variation of Epicephala moth assemblages, immature fruits of K. microcarpa were collected from 16 sites of four provinces in China (Table 1) to rear larvae and adult moths. The fruits were placed in plastic food containers with wet tissue in the bottom to keep a certain air humidity. We recorded number and sexes of reared moths, then randomly dissected more than 10 moths (with equal numbers of males and females) from each site to identify the moth species based on morphology (Table 1). Additionally, forewing length (proxy for body size) and genitalia traits (Figures S2 and S3, male moth: saccus length, vinculum width, costa length, tegument length, phallus length; Figure 3 and Figure S3, female moth: ovipositor length, lamella antevaginalis length, apophysis anterioris length, apophysis posterioris length, keel length) were measured for each F I G U R E 1 Habitat (a), inflorescences and immature fruits (b), female flower (c), and male flower (d) of Kirganelia microcarpa; pollination behavior of the moth Epicephala microcarpa, in which a female moth inserts her proboscis into the stylar pit (e), and oviposition behavior in which she laterally cuts through the ovary wall (f); pollination (g) and oviposition (h) behavior of the moth E. tertiaria, in which a female moth inserts her proboscis and then her ovipositor into the stylar pit; egg (i) and larva of Epicephala sp. in stylar pit of the flower (white arrows). dissected moth. Both male and female genitalia were removed and stained with Eosin dye solution before measurements.
To investigate plant trait variation among populations, 30 leaves, 30 male flowers, and 30 female flowers were randomly sampled from 3 to 10 individuals (Table 1) for each site to measure leaf length, leaf width, stamen length, sepal length, stylar pit depth (distance from stigma to the placenta), and ovary wall thickness ( Figure S4).

| Data analyses
To examine whether ovipositor traits are correlated with other body traits, we conducted bivariate correlation analyses between forewing length (proxy for body size) and both male (phallus length) and female genitalia traits (ovipositor length, keel length). To estimate the degree of matching between ovipositor length and flower traits, we calculated mismatch index (MMI) with the equation MMI = (OL − FT)/ FT, where OL represents moth ovipositor length and FT represents flower traits (stylar pit depth or ovary wall thickness). MMI = 0 means an exact match, −1 < MMI < 0 means the ovipositor is shorter than the flower traits, while 0 < MMI < 1 means the ovipositor is longer than the flower traits. The absolute value of MMI represents the degree of trait mismatching. To examine the trait matching between flowers and moths, generalized linear model analyses (GLMs) with a normal distribution and an identity function were carried out to compare the differences between stylar pit depth, ovary wall thickness, and ovipositor length of three Epicephala moths and to compare the differences of keel length among three moth species.
To examine whether ovipositor traits match flower traits among different sites, bivariate correlation analyses were conducted between female flower traits (stylar pit depth and ovary wall thickness) and moth ovipositor traits (ovipositor length and keel length) for both E. microcarpa and E. tertiaria.
To examine whether flower traits and insect traits respond to different moth assemblages, pairwise contrast of GLMs with a normal distribution and an identity function were conducted to compare the differences of stylar pit depth and ovary wall thickness with moth assemblage as a treatment variable, that is, K. microcarpa populations associated with E. microcarpa alone (Em), in which E. microcarpa and E. laeviclada (Em-l) coexisted, in which E. microcarpa and E. tertiaria (Em-t) coexisted, in which E. microcarpa, E. tertiaria, and E. laeviclada (Em-t-l) coexisted, and in which E. tertiaria and E. laeviclada (Et-l) coexisted. Additionally, pairwise contrast of GLMs with a normal distribution and an identity function were conducted to compare differences of ovipositor length and keel length of E. microcarpa moths which coexisted with E. tertiaria or/and E. laeviclada, and the differences in ovipositor length and keel length of E. tertiaria moths which coexisted with E. microcarpa or/and E. laeviclada, respectively.
All analyses were conducted in SPSS v. 22.0 (IBM Inc.), all histogram, and scattergram were made in Origin v. 9.0 (OriginLab Inc.), and all data were logarithmically transformed to be normally distributed before analysis.

F I G U R E 2
Morphology of head and proboscises of three species of Epicephala moths under a stereo microscope. Female moth (a) of the pollinator E. microcarpa with dense hairs on proboscis (e, black arrow); female moth (b) of the pollinator E. tertiaria with dense hairs on proboscis (f, black arrow); female moth (c) of cheater E. laeviclada with few short hairs on proboscis (g); male moth (d) of pollinator E. microcarpa without hairs on proboscis (h).
TA B L E 1 Longitude, latitude, moth species assemblages, number of moths reared, number of moths dissected for species identification, number of plant individuals from which moths were reared, and number of plant individuals from which trait measurements were taken, for all 16 sites in this study.

| Pollination and oviposition behaviors
The

| Spatial pattern of Epicephala moth coexistence
Our field observations and rearing results showed that multiple Epicephala moth species coexisted at all studied populations except Diaoluoshan ( Figure 4; Table 1). Specifically, all three moth species coexisted (Em-t-l) at six populations, E. microcarpa and E. tertiaria  Table 1). The cheater E. laeviclada occurred at 10 out of 16 populations and always coexisted with pollinators ( Figure 4; Table 1), suggesting geographic variation in moth assemblages associated with K. microcarpa.

| Epicephala ovipositor morphology associated with floral traits
Correlation analyses showed that phallus length was significantly positively correlated with forewing length in both E. microcarpa and E. tertiaria male moths (  (Figure 5a), while the latter two species did not show significant difference from each other (p = .766; Figure 5a), indicating that ovipositor length differed among moth species with different oviposition behaviors. Although comparison analyses showed significant differences between female flower traits (stylar pit depth or ovary wall thickness) and ovipositor length (Figure 5a), the mismatch index results suggested that ovipositor length of E. microcarpa and E. laeviclada more closely matched ovary wall thickness ( at species level. Similarly, these patterns were seen globally as well as in each studied population ( Figure 6; Table 5) and among populations with different moth assemblages ( Figure 7).   systems where the match between plant and insect traits will affect pollen deposition and plant fitness (Muchhala & Thomson, 2009).

| Trait matching in situations with multiple partners
Similarly, trait matching between ovipositor length and floral traits should be favored for oviposition behaviors in specialized seedpredating pollination systems, although this may be due either to mutualistic or antagonistic coevolution (Godsoe et al., 2010;Yang & Li, 2018). In this study, three Epicephala moth species laid eggs in female flowers using diverse oviposition behaviors, and our results suggested a match between a longer ovipositor and flower stylar pit depth in E. tertiaria performing stylar pit oviposition (Figure 5a,b; Table 4), and a match between short ovipositors and ovary wall thickness in E. microcarpa and E. laeviclada performing locular oviposition (Figure 5a,b; Table 4). These results support the hypothesis that moth ovipositor traits are adapted to specific flower traits, depending on the oviposition behavior of the moth species. Additionally, our results suggested trait matching between floral traits and ovipositor length for both pollinators (E. microcarpa and E. tertiaria) and cheater (E. laeviclada) at species level, which is consistent with the positive correlations between style length/wall width and pollinator/non-pollinator ovipositor length in fig-fig wasp mutualism (Weiblen, 2004).

F I G U R E 9
Comparisons of stylar pit depth (a; mean ± SE) and ovary wall thickness (

TA B L E 4
Mismatch index (MMI) between ovipositor length of three Epicephala moth species and female flower traits (stylar pit depth and ovary wall thickness). MMI = 0 means an exact match, MMI < 0 means the ovipositor is shorter than the flower trait, and MMI > 0 means the ovipositor is longer than the flower trait. The absolute value of MMI represents the degree of trait mismatch.

Mismatch index (MMI)
Ovipositor length In other studies, functional traits of interacting partners, such as nectar spur length of flowers and pollinator proboscis length in pollination mutualisms (Darwin, 1862), or cone structure and bill size in seed dispersal interactions (Benkman et al., 2003), have been shown to be strongly correlated among different sites due to stabilizing selection from a primary partner species, which results in a coevolutionary arms race. Empirical evidence from the pollination mutualism between long-tongued flies and the plants Zaluzianskya microsiphon (Scrophulariaceae) and Lapeirousia anceps (Iridaceae) supports this hypothesis (Anderson & Johnson, 2008;Pauw et al., 2009). However, whether such arms races can explain trait matching when partners are part of multi-species assemblages with various relevant trait values is less clear (Haber & Frankie, 1989;Harder, 1985). In a study of an oil-producing flower and three species of oil-collecting bees,  (Tables S1 and S2). Additionally, our results indicated that stylar pit depth was less in populations with E. tertiaria as the primary pollinator and greater in populations with E. microcarpa (Table 6), suggesting that plants may be responding to pollinator oviposition behaviors, although there were no significant correlations between ovipositor length and floral traits among different populations (Table 2). Since all three moths exhibited close trait matching of ovipositor length to floral traits at species level, the lack of covariation between functional traits at the population level might reflect the effects of selection from multiple partners, as well as amongpopulation gene flow (Yoder et al., 2013).

Hollens
The geographic mosaic theory of coevolution (GMTC; Thompson, 2005) predicts that at the metapopulation level, interacting partners may in some populations show both trait matching (hot spots) and trait mismatching (cold spots) (Gomulkiewicz et al., 2000;Thompson, 1999). Although we know that evaluating the selection is important for testing GMTC, data are not available for us to do

| Trait variations in response to different interacting partners
In mutualisms, interacting partners are expected to evolve mechanisms to limit excessive mutual exploitation, thus maintaining the mutualism (Pellmyr & Huth, 1994). Partner choice, host sanctions, and partner fidelity feedback are among the mechanisms that have been hypothesized to function to maintain mutualism (Bull & Rice, 1991;Leigh, 2010;Weyl et al., 2010). In this study, we examined how local moth species assemblages affect the covariation of functional traits in both female flowers and moth ovipositors, which may have implications for mutualism stability. Our results indicate TA B L E 6 Effects of different Epicephala moths and behaviors on floral trait (stylar pit depth and ovary wall thickness) variations. Bold values indicate significant differences at p < .05 under generalized linear model analyses. The letters in brackets indicate moth pollinator (P) or cheater (C). The symbol "↓" indicates decreasing trends, "↑" indicates increasing trends, and "−" indicates no significant variation. showed that ovary wall thickness significantly increased when the cheater E. laeviclada existed in the populations (Figure 9d; Table 6).
Similarly, this increase also occurred when the pollinator E. microcarpa existed (Figure 9d; Table 6), which means that the plants might increase ovary wall thickness to defend against locular oviposition regardless of the ecological role of the insects (pollinator or cheater).
However, the ovary wall thickness of flowers in populations with all three moths showed no significant difference (p = .173) with that of flowers from populations with only E. microcarpa and E. tertiaria (Emt; Table 6) and was significantly thinner (p < .001) than that of flowers from populations with only E. microcarpa and E. laeviclada (Em-l; Figure 9d), for reasons that remain unclear. In addition, ovary wall thickness was always higher in the presence of E. microcarpa relative to when E. microcarpa was absent (Figure 9d), indicating that E. microcarpa is probably the main factor determining variation in ovary wall thickness. This pattern is probably due to the high abundance of E. microcarpa in most plant populations (Table 1).
Moreover, stylar pit depth decreased in response to the existence of E. tertiaria which exhibited pollination and oviposition via the stylar pit and increased in response to E. microcarpa pollinating via the stylar pit ( Figure 9d; Table 6). This is interesting, since E. tertiaria larvae do also consume host seeds, and so we might expect K. microcarpa to evolve to reduce E. tertiaria oviposition as well.
However, we still do not know whether the variation in stylar pit depth is a response to oviposition or pollination, since we lack of proboscis length data. but also in oviposition parasitism.

| Mutualism and phenotypic differentiation
Ovipositor morphology is critical for host plant use and copulation, which may then affect plant-insect interactions and reproductive isolation (Althoff, 2014;Joy & Crespi, 2007). In the evolution of specialized yucca-yucca moth interactions, ovipositor length and morphology differentiated, suggesting adaptation to different oviposition behaviors, which may then lead to reproductive isolation and speciation (Althoff, 2014;Althoff et al., 2006). In this study, the pollinator E. microcarpa and cheater E. laeviclada exhibited shorter ovipositors and different oviposition behavior than the pollinator E. tertiaria, and the phallus length was correspondingly shorter in the former two species than that in E. tertiaria (Table 4), similar to the association between morphological differentiation and reproductive isolation in yucca moths (Althoff, 2014).
However, it remains unclear how E. microcarpa and E. laeviclada (which have the same ovipositor length and locular oviposition in female moths, and the same phallus length in male moths) maintained reproductive isolation during the process of speciation and whether they have interspecific gene flow today. Either a period of allopatric isolation during the process of speciation or a host shift by one moth species from another species of Kirganelia to K. microcarpa seems likely; both have been demonstrated in other leafflower-leafflower moth systems (Hembry et al., 2013;Luo et al., 2017). Additionally, many genitalia traits such as serrature length, apophysis anterioris length, ova length, apophysis posterioris length in female moths and saccus length, costa length, tegumen length in male moths showed significant differences between these two species (Table S3), which might affect the mating process and reproductive isolation. Since these two species have the same oviposition niche, it seems likely that E. microcarpa and E. laeviclada compete strongly with each other (Hardin, 1960). If E. microcarpa is somehow the superior competitor, that might explain the low frequency of E. laeviclada in all populations expect Zhaoqing (Table 1), and contribute to maintaining the mutualism.
In this study, we did not estimate seed costs resulting from different Epicephala moth species, so we could not discuss to what extent the presence of the cheater will lead to mutualism breakdown.
As our study focuses on one plant species and trait matching be- Conceptualization (equal); funding acquisition (lead); investigation (supporting); resources (lead); supervision (lead).

ACK N OWLED G M ENTS
We thank Lian-Jie Zhang and You-Heng Wu for help with the fieldwork and David Althoff for discussion and providing comments on the manuscript.

FU N D I N G I N FO R M ATI O N
This work was supported by the National Natural Science Foundation of China (No. 32270242, 31370268) to S.X.L.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
No data are available.